胶原蛋白作为医用生物材料的应用

胶原蛋白是一类具有活跃生物功能的细胞外间质成分,占人体总蛋白质总量的1/3。胶原蛋白是组织的支持物和填充物,也主动参与了细胞迁移、分化及增殖,并与创伤修复及胚胎发育有关。以胶原蛋白为原料制备的生物替代物(如人造皮肤等)必然大大优于非生物材料的同类产品。目前,大量不同性能及用途的胶原蛋白医用材料不断涌现,有些已结束实验室及动物实验阶段,大批量投入临床应用。     

微米级羰基铁粉磁性骨水泥的制备与表征

背景：目前有日本学者提出采用磁性骨水泥治疗肿瘤的骨转移,然而现行磁性骨水泥添加的均为纳米级磁流体,将微米级羰基铁粉添入骨水泥的研究尚未见报道。 目的：制备含不同比例微米级羰基铁粉的磁性骨水泥,并按ISO 5833标准测量相关指标及其磁性与体外升温情况。 方法：将微米级羰基铁粉与聚甲基丙烯酸甲酯骨水泥混合制备成含羰基铁粉质量分数分别为0%,20%,30%,40%,50%的磁性骨水泥。将以上5组材料按ISO 5833标准测定凝固时间、聚合温度、抗压强度。并采用振动样品磁强计测定各组磁性及在交变磁场下的升温情况。 结果与结论：随着羰基铁粉含量的增加,磁性聚甲基丙烯酸甲酯骨水泥的凝固时间有所延长。各组骨水泥的最高聚合温度均在65-70 ℃之间,并未随羰基铁粉含量的增加而改变,仅聚合温度最高点出现的时间随羰基铁粉含量提高而后延。各组骨水泥的抗压能力均大于60 MPa,但只有聚甲基丙烯酸甲酯骨水泥的抗压能力＞70 MPa,符合IOS 5833标准要求。各组磁性骨水泥的磁饱和强度随羰基铁粉含量的提高而提高。在交变磁场下,磁性骨水泥的升温速率与磁场强度和羰基铁粉含量呈正相关。     

医用热像图的理论基础和临床应用

简要介绍了有关医用红外热像图的基础理论和在临床应用概况。按物理学的观点认为 ,人体是一种自然的红外辐射源 ,测量人体体温是临床诊断的一种重要指标。根据普朗克定律得知 ,量子能量为E =hf,又根据维恩定律计算 ,人体红外线辐射波长在 5～ 5 0微米之间 ,最大波长为λmax=9微米 ,反射率为 1。本文也概要的介绍了当今医用红外热图在临床应用的进展。劳森博士首先发现了乳腺癌病变表面皮肤温度明显高于正常乳房皮肤温度。从那时起 ,医用红外热像图技术在临床应用上有了快速发展 ,在世界范围内成立了一些专业组织并出版了有关医用红外热像图杂志。本文也概要的介绍了当今医用红外热像图在临床应用的进展。     

胶原蛋白海绵交联工艺的优化

背景：胶原蛋白材料具有良好的生物相容性和生物可降解性,但在临床应用过程中也暴露出了机械强度低、耐降解性能差等问题。大量研究报道,通过适当的交联可以改善胶原蛋白材料的缺陷,调控其多孔网络结构、溶胀性和降解性。 目的：优化胶原蛋白海绵的碳化二亚胺交联工艺,探讨其最佳工艺条件。 方法：利用碳化二亚胺对胶原蛋白海绵进行交联改性,得到具有疏松、多孔网络结构的胶原蛋白海绵,同时采用正交实验对交联工艺进行优化,单因素中选择碳化二亚胺浓度(5,10,20,30,40,50,60,70,80,90,100 mmol/L)、交联时间(2,4,6,8,12,16,20,24 h)及交联温度(5,10,15,20,25,30,35 ℃)为实验因子,以孔径、孔隙率、吸水性、降解率来筛选胶原蛋白海绵交联的最佳工艺。 结果与结论：当碳化二亚胺浓度为50 mmol/L、交联温度为20 ℃、交联时间为6 h时,胶原蛋白海绵的各项性能最为优越,为最优工艺条件,其中平均孔径大小为105 μm,孔隙率为79.45%,吸水率为287.14%,降解率最优为15.04%(2 d)。表明通过对胶原蛋白海绵的交联改性,极大提高了海绵的吸水性能和耐降解性能。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

酸性成纤维细胞生长因子/胶原蛋白复合海绵的研制及其组织相容性

我们以牛跟腱和酸性成纤维细胞生长因子(acidic fibroblast growth factor,aFGF)为原料,研制了一种新型的创伤敷料--aFGF/胶原蛋白复合海绵,并检测了其物理性能和组织相容性,特别是在血液相容性方面,研究其作为医用生物材料的安全性.结果显示:制备的高剂量和低剂量aFGF/胶原蛋白复合海绵的急性毒性试验、刺激性试验结果均为阴性;复钙试验表明aFGF/胶原蛋白复合海绵复钙时间较长,具有较好的抗凝特性;溶血试验表明复合海绵对红细胞的破坏很小,符合生物材料的溶血性要求;血小板黏附试验表明aFGF/胶原蛋白复合海绵的血小板黏附量较少且未被激活,对血小板没有明显的破坏作用.结果表明,aFGF/胶原蛋白复合海绵具有较好的组织相容性,具备临床应用的可能性.     

鱿鱼软骨Ⅱ型胶原蛋白提取方法及结构分析

目的从鱿鱼软骨中分离纯化非变性II型胶原蛋白,并对其进行结构表征。方法将鱿鱼软骨冻干后粉碎成200目软骨粉,用4%(w/w)Na OH溶液去除多糖、0.5 M EDTA溶液脱灰对软骨进行前处理,采用胃蛋白酶水解提取,Na Cl盐析用纯水透析后冷冻干燥得到II型胶原蛋白样品。通过氨基酸组成分析、SDS-PAGE电泳、紫外吸收光谱、电镜扫描等对制备的样品鉴定。结果制备的II型胶原蛋白具有3条α1链,分子量112 k Da,在224.5 nm波长处有最大紫外吸收,具有三螺旋结构特征。     

70 mer长寡核苷酸探针芯片制备、标记方法及杂交条件的优化

目的优化长片段寡核苷酸芯片制备、探针标记及杂交条件。方法通过直接标记方法优化芯片的点样浓度、点样后的固定方法。探索两种靶基因标记方法,并优化杂交液及各种杂交条件。结果采用70mer寡核苷酸探针,点样浓度在10μmol/L时就可获得满意的荧光强度。探针点样后37℃水合,然后在3600mJ条件下进行紫外交联固定效果最好。样品采用生物素间接标记较CY3直接标记信号强度强,成本相对低廉。优化出的杂交液成分:50%甲酰胺、5×SSC、0.5%SDS,杂交时间为8h,杂交温度为65℃。结论通过此试验优化了制备病原体长探针寡核苷酸芯片的制备方法,样品标记策略和杂交液及杂交温度,为下一步制备多种病原体的寡核苷酸芯片检测系统奠定了基础。     

双靶向配体化力达霉素DTLL的制备优化和稳定性研究

目的通过对具有高效抗肿瘤活性的类抗体偶联药物—--双靶向配体化力达霉素(DTLL)制备过程的优化,获得纯度高和活性强的DTLL产品;探讨影响DTLL稳定性的主要因素,为临床申报和工业化生产提供依据。方法分别对DTLL前体(DTLP)蛋白表达载体、宿主菌株、表达温度、诱导剂终浓度、菌体密度和诱导时间进行优化。以高压破碎法提取可溶性目的蛋白,采用Ni+亲和与分子筛层析两步纯化,得到高纯度DTLP,再与力达霉素发色团组装获得DTLL。采用HPLC、MTS、ELISA法进行稳定性试验,分别监测DTLL冻干样品的蛋白纯度、发色团含量、细胞毒和亲和力活性的变化。结果经过发酵提取工艺优化,每升发酵液所得DTLP蛋白约为22 mg,是优化前每升9 mg的2.4倍,其蛋白纯度达到99.7%;DTLL组装效率达到68%;稳定性试验结果显示,DTLL冻干样品对光照敏感,在–80℃低温避光环境下可稳定保存。结论成功优化了双靶向配体化力达霉素DTLL的制备工艺,提升了产品纯度和产率,为其后续生产和临床申报提供了实验基础,也为其他抗体偶联药物的制备提供了较成熟完善的技术平台。     

bFGF胶原海绵的制备及其组织相容性评价

目的 以重组人碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)、I型胶原蛋白为主要原料，研制一种新型的创伤敷料，并对其组织相容性进行检测，评价其作为医用生物材料的安全性。方法 制备高纯度的I型胶原蛋白溶液，加入bFGF后冷冻干燥成为海绵体，进行组织相容性试验，包括急性毒性试验、刺激性试验、过敏性试验和溶血性试验。结果 制备的bFGF胶原海绵的急性毒性试验、刺激性试验、过敏性试验和溶血性试验结果均呈阴性。结论 所研制的bFGF胶原海绵具有良好的生物相容性，无毒副作用，是可以安全使用的医用生物材料。     

胶原蛋白的市场前景

胶原蛋白是人体内含量最多、分布最广的蛋白质，是一种与组织和器官功能密切相关的功能性蛋白。胶原蛋白主要分布在人体的结缔组织中，对人体皮肤，血管，骨骼，筋腱和软骨的形成以及维持其韧性和弹性都十分重要。     

The Application of Recombinant Human Collagen in Tissue Engineering

Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.     

The structural basis of cell-mediated immunological reactions of collagen: The species specificity of the cutaneous delayed hypersensitivity reaction

Guinea-pigs were sensitized for delayed hypersensitivity skin reactions with native rat, rabbit, fish, or human collagen or with (L-Pro-Gly-L-Pro)_n, mixed with Freund's complete adjuvant (FCA). Mammalian collagens were more immunogenic than fish collagen and the synthetic, collagen-like polymer. The terminal, non-helical portions of calf and rat collagen were not required for expression of species specific cell-mediated reactions of these collagens. Although the animals could readily distinguish between mammalian collagens on one hand and fish collagen and (L-Pro-Gly-L-Pro)_n on the other, the discriminatory capacity was best expressed among each of the two groups of antigens.     

Experimental pyelonephritis in the cat: 3. Collagen alterations in renal fibrosis

Chronic pyelonephritis was induced in young adult cats by the intravenous injection of a human or a feline strain of Escherichia coli after ligation of one ureter for 24 or 48 h. In the 3 cats infected with the feline strain, scarred kidneys from the obstructed side were removed at necropsy 3, 4 and 5 months later. Collagen was extracted from pyelonephritic and normal kidney tissue with dilute acetic acid and limited proteolysis with pepsin. Scarred kidneys gave higher yields of both acid-soluble collagen (normal = 0.57 +/- 0.12 mg per g tissue; scarred = 0.88 +/- 0.10 mg per g tissue) and pepsin-solubilized collagen (normal = 9.69 +/- 1.79 mg per g tissue; scarred = 20.02 +/- 2.84 mg per g tissue). There was no significant increase in the collagen yield from the kidneys of the 2 cats in which mild focal lesions were found 14 and 16 months after infection with the human strain of E. coli. Pepsin released collagens were separated by fractional salt precipitation and identified by agarose gel chromatography and polyacrylamide gel electrophoresis. Normal kidney was shown to contain collagen of Types I, IV and V (AB). The Type IV collagen extracted consisted of a mixture of 4 major pepsin-resistant chains of apparent molecular weights of 150 000, 115 000, 85 000 and 60 000. The collagen extracted from scarred kidneys was predominantly Type I, only trace amounts of Type IV and V components being present. These findings suggest that basement membrane collagens of the kidney are selectively degraded during the atrophy and scarring of chronic feline pyelonephritis and are preferentially replaced by interstitial Type I collagen.     

Age-related changes in the proportion of types I and III collagen

Previous studies of the age-related changes in interstitial collagens, have suggested that the proportion of type III collagen compared with type I decreases with age. In this study collagen concentration and the proportion of types were measured in heart, lung and skin of male Lewis rats aged between 1 day and 2 years. Collagen concentration, based on hydroxyproline levels, increased in all tissues up until 6 months of age, thereafter it increased in heart and lung, yet decreased in skin. The relative proportions of types I and III collagen were assessed after cyanogen bromide digestion of the tissue and separation of the resultant peptides by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At 2 weeks of age collagen type III represented about one-third of types I and III in all tissues. After this age the proportion of type III increased up to 1 year in both heart (53.9 +/- 1.8%) and lung (47.7 +/- 2.8%), whereas it decreased in skin (18.6 +/- 1.5%). These data show that there are age-related changes in both collagen concentration and the relative proportions of types I and III collagen, but that the direction of these changes differs between tissues.     

Vascular collagens. General review

The analysis of vascular collagens proved difficult due to high insolubility. In the seventies, compositional studies concerned a fraction of 30% of total collagen. Type I collagen, far less soluble than type III, was not fully extracted, resulting in an overevaluation of type III collagen. When the proportions of collagens are measured on the totality of the material, type I collagen represents 60%, type III, 30% and the remaining 10% are constituted by type V and minor collagens. Atheromatous plaques contain a little more collagen than normal arteries and the proportion of type I remains 60%. Whereas several experiments demonstrated that slices of atheromatous arteries in vitro synthesize more collagen than slices of normal arteries, the possible mechanism of interaction between plasma lipoproteins (normal or abnormal) and collagen metabolism remains unknown. Vein and capillary collagens have been rarely studied. Basement membrane collagen does not seem to be increased in quantity in the thickened basement membrane of diabetic patients capillaries.     

机械压应力通过TGF-β1通路治疗增生性瘢痕的分子机制研究

目的　通过机械压应力系统干预增生性瘢痕成纤维细胞,研究皮肤创伤后增生性瘢痕组织中TGF-β1通路中TGF-β1受体、smads和胶原蛋白的差异性表达,探究压应力治疗增生性瘢痕的分子机制。 方法　体外培养增生性瘢痕细胞,应用RT-PCR方法比较机械性压应力干预组和空白对照组增生性瘢痕成纤维细胞内Smad3、Smad7和胶原蛋白表达差异。 结果　根据RT-PCR结果,增生性瘢痕成纤维细胞系中在机械性压应力干预后,TGF-β1受体表达量增高,Smad3、 Smad7、CollagenⅠ表达量下降,差异有统计学意义（P<0.05）,collagenⅢ表达两组差异无统计学意义（P>0.05）。 结论　机械压应力治疗增生性瘢痕可通过TGF-β1通路中抑制Smad3的表达从而促进胶原蛋白Ⅰ的降解。     

Use of an in vitro model of tissue-engineered skin to investigate the mechanism of skin graft contraction

Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinical problem. In this study we used a tissue-engineered model of human skin, based on sterilized human adult dermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as a model to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagen synthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesis and degradation were determined using immunoassay techniques. The results show that skin contraction was not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but was related to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the ability of cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyl oxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary, this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possible pharmacologic targets for prevention of graft contracture in vivo.     

The structural basis of cell-mediated immunological reactions of collagen: Reactivity of separated α-chains of calf and rat collagen in cutaneous delayed hypersensitivity reactions

Denatured calf and rat skin collagen and α1- and α2-chains from these collagens were tested for their capacity to sensitize guinea-pigs for the cutaneous delayed hypersensitivity reaction and to elicit this reaction in sensitized animals. Alpha-chains from neutral salt extracted and from urea extracted collagen were also compared. All preparations were fully active as sensitizers as well as elicitors of the skin reaction. In spite of extensive cross-reactions, significant differences between α1- and α2-chains of a given species as well as between corresponding chains of different species could be detected. Differences between chains from neutral salt extracted collagen and from urea extracted collagen were also revealed.     

应用胶原海绵构建组织工程皮肤的实验研究

目的探讨以胶原海绵为支架材料构建组织工程皮肤的方法。方法体外分离、培养、鉴定角朊细胞和成纤维细胞,制备胶原海绵作为组织工程支架材料;在成功构建人工真皮的基础上,种植表皮细胞,构建人工复合皮肤,通过大体观察、HE染色与免疫组织化学进行组织学检测。结果制备的胶原海绵孔径平均100~150μm,孔隙率89%,组织相容性良好;以胶原海绵构建的皮肤替代物免疫组织化学染色显示Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,HE染色可见均具有表皮和真皮双层结构,在形态结构上与正常皮肤相似。结论培养的人角朊细胞和成纤维细胞种植于胶原海绵支架上培养可构建出具有类似天然皮肤结构的组织工程皮肤。     

瘢痕组织中胶原蛋白提取及纯化研究

目的 优化实验条件,建立提取瘢痕胶原蛋白的可靠方法。方法 选择2015年1月~2016年12月本院20例手术整形切除增生性瘢痕的标本进行研究,用胃蛋白酶的消化方式、用盐酸溶解法提取瘢痕胶原,用氯化钠盐析法制成胶原蛋白膜,用吸收光谱分析、氨基酸成分分析对胶原蛋白进行鉴定。结果 20例胶原标本提取胶原蛋白,通过氨基酸成分分析甘氨酸占30%以上,吸收光谱分析在230 nm处有吸收波峰,证实提取物为来自皮肤瘢痕组织Ⅰ、Ⅲ型胶原蛋白。结论 用酶消化、酸溶解、盐析法提取瘢痕胶原蛋白,方法可靠,操作简单,提取胶原蛋白纯度高,符合Ⅰ、Ⅲ型胶原蛋白特征,制成胶原蛋白膜为进一步研究瘢痕提供了生物学模型。